Insights into a viral motor: the structure of the HK97 packaging termination assembly

Abstract Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.


INTRODUCTION
Encapsulation of the genome r epr esents a key step in viral assembly and life cycle. For double stranded (ds) DNA bacteriophage, the genome is packaged into a preformed protein shell, or capsid, thr ough the dodecameric portal pr otein, which acts as a door ( 1 ). Packaging often produces expansion of the capsid from an immature form, known as a prohead, to a larger, fully mature state.
Although mechanisms of packaging are broadly conserved among the dsDNA phages, further differentiations can be highlighted based on processing of the viral DNA at the initiation and termination of packaging. Cos viruses, such as , P2, and HK97 --the subject of this paper --package exact genome-length DNA units ( 18 , 19 ), separated by consecuti v e cos (cohesi v e) sites in a genome concatemer ( 20 ). For pac viruses, including P22, P1, SPP1 and T4, packaging is initiated from a pac (packaging) site within the genome (21)(22)(23) and terminates in response to 'headful' pr essur e within the prohead, leading to encapsulated genomes varying in length from ∼103 to 110% ( 24 ). Finally, 29-like phages produce unit-length DNA genome copies, where each 5 end is covalently bound to viral protein gp3, which facilitates packaging ( 25 ).
Portal proteins display highly di v ergent sequences and molecular weights, but in situ within the viral capsid, consistently present as dodecameric rings (26)(27)(28). The monomeric portal protein structures share conserved protein folds and domain arrangement comprising the clip, stem, wing and crown domains. Positioned at the base of the portal protein, the clip domain interacts with the large terminase motor during assembly, and later the adaptor proteins for tail attachment ( 26 ). The stem contains the 'tunnel' ␣-helix that lines the internal channel with se v eral negati v ely charged residues ( 29 ). The most structurally di v ergent domain, the wing, coordinates contact with the capsid proteins (26)(27)(28). Lastly the crown, exposed at the inner surface of the capsid, interacts with packaged DNA ( 30 ).
The portal protein displays remar kab le plasticity, proposed to facilitate symmetry-mismatching interactions with the capsid and terminase. This flexibility also permits propagation of pr essur e changes within the capsid to the terminase ( 31 ), in order to induce packaging termination. Indeed, the DNA in mature phage P22, is spooled tightly around the portal in an arrangement which appears to be incompatible with the procapsid portal form ( 31 ). Meanwhile several discrete mutations within the P22 and λ portal cores produce over-packing phenotypes ( 32 , 33 ), indicating unsuccessful ter mination. Confor mational changes within the portal protein during DN A packaging a ppear to be a conserved feature among dsDNA viruses, with marked variability between procapsid and mature head portal structures for T7, 29, p22 and P23-45 ( 30 , 31 , 34-38 ).
Much mechanistic understanding of the large terminase motor has been drawn from single molecule optical tweezer studies on the phage 29. The 29 large terminase mechano-chemical cycle has been shown to alternate between two phases. During the dwell phase ATP binds cooperati v ely to each subunit around the ATPase ring, and the DNA substrate remains stationary. During the burst phase, DNA translocation into the capsid occurs in four subsequent 2.5 base pair steps, corresponding to four ATP hydrolysis e v ents ( 39 ). A cryo-EM reconstruction of the intact 29 packaging motor, comprising the capsid, pRNA, large terminase and DNA, re v ealed the fiv e AT-Pase domains, in a 'cracked' helical conformation, stabilised by ATP γ S ( 40 ).This conformation contrasts planar structures seen for the apo 29 ATPase ( 41 , 42 ) and the ADPbound form of closely related phage ascc-28 large terminase ( 43 ). The transition between the cracked helical and planar states likely r epr esents the burst phase, which has inspired a unique DNA translocation model ( 43 ). Howe v er, other terminase-packaging systems may well adopt mechanisms different to 29, w hich unusuall y utilises pRNA and does not r equir e small terminase protein ( 42 , 44 ).
Whilst a wealth of structural data comprising individual packaging proteins has emerged for dsDNA viruses, the field is only starting to move towards analysing complete packaging systems. For bacteriophage HK97, the focus of present study, structural information is so far available only for individual components including large and small terminase proteins ( 45 ) and prohead II (the immature prohead sta te a t packaging initia tion) ( 46 ). The X-ray structure of monomeric large terminase re v ealed the classical ATPase and nuclease domains adjoined by a short linker ( 45 , 47 ). In common with most other phage, the small terminase contains 9 subunits arranged in a circular structure exposing Nterminal HTH motifs around the outside of the oligomerisation core of the molecule, where they form a positi v ely charged rim ( 36 , 38 ).
The cryo-EM structure of the acti v e HK97 packaging machinery pr esented her e, in combination with pr evious wor k estab lishing a functional DNA packaging assay ( 45 ), begins to shed light on a number of unanswered questions for cos phage: (i) how do these viruses overcome the symmetry mismatch between the 12-subunit portal and pentameric large terminase, (ii) does small terminase remain engaged with the motor throughout packaging and (iii) what is the mechanism of coordinated ATP hydrolysis around the large terminase ring and how are the chemical e v ents of ATP hydrolysis coupled to mechanical translocation of DNA.

Packaging assays
Individual packaging protein components of the packaging assays wer e expr essed and purified as described ( 45 ). The DNA substrate used r epr esented a linearised pUC18 plasmid with an engineered cos site from -312 to +472 of the cleavage sites. Limiting the packaging time from 30 to 2 min significantly improved large terminase retention, and the addition of DNAase1 to the final sample improved the background signal.

Data collection
R 3.5 / 1 Quantifoil, with 2 nm Ultrathin Carbon, 200 mesh copper grids were glo w dischar ged for 60 s at 15 mA in a PELCO easiGlow ™. A 3 l sample was prepped immediately prior to vitrifying using the Vitribot IV, with blot force -5 and blot time 2 s ( 54 ). This grid was subject to a 72-h data collection at the UK National eBIC (Electron Bio-Imaging Centre) at the Diamond Light Source (Harwell Science and Innovation Campus) on an FEI Titan Krios instrument using a K3 detector. The data collection parameters are summarised in Supplementary Table S1.

Reconstruction of the prohead / portal / motor complex (Fig S1)
Data were processed in RELION 3.1 ( 48 ). Motion correction was performed using MotionCor2 ( 49 , 50 ) and corrected micro gra phs were then subject to CTF estimation using CTFFIND 4 ( 51 ). Optimised picking parameters were achie v ed using Topaz, as an external RELION job ( 52 ). A total of 352 600 particles were picked and subject to 2D classification. The best classes were selected, and duplicates removed, leaving 82 279 particles for icosahedral refinement. This map was subject to post processing and CTF refinement before a second round of refinement ( 53 ) (Supplementary Figure S1A). The resolution of the final post processed reconstruction is 3.06 Å (FSC = 0.143).
During icosahedral refinement signal from asymmetric features of the virus is averaged out evenly over the map. Thus, in order to model the portal and motor, the icosahedral 3D reconstruction of the combined data sets was first subject to RELION symmetry expansion ( 53 )(in symmetry point group I3), which generates a 60-fold increased set of particles. Knowing the location of the vertices, we could determine ne w e xtraction coor dinates ( 54 ) for re-e xtraction of each v erte x whilst binning to 5.2 Å / pixel. This produced 60 subparticles per capsid particle. Subparticles were subject to 3D classification without alignment to separate subparticles containing portal and motor signal, from pentameric ca psomers. Initiall y a cylindrical mask, created using relion helix toolbox ( 55 ) with a soft edge of 2 pixels and extension of 2 pixels, was used to mask out contributing signal from the capsid, centred on the expected position for the portal and motor. One clear portal-containing class emerged, which was used as a template for a tighter mask applied to the original subparticle set for further classification.
To reconstruct the portal protein structure, particles from the portal-motor class were selected and subjected to 3D classification without performing image alignment. The classes displaying the most defined secondary structure were selected and re-extracted into a smaller box encompassing only the portal (1.34 Å / pixel). These 57279 particles wer e r efined using C12 symmetry. The 3D map was then subject to postprocessing reaching 2.98 Å resolution (FSC = 0.143) and local resolution estimation to calculate r esolution within differ ent local sections of the map (Supplementary Figure S1B).
The motor signal was isolated by re-extraction of particles at the portal v erte x into a smaller box size encompassing the motor density only. These were subject to 3D classification without performing image alignment, and classes displaying clear individual subunits and domains were subject to a second classification into a single class. This was used as a template for a mask used for particle subtraction. This class contained just 17584 particles. Subtracted particles were subsequently subject to refinement with local angular searches (1. The run data.star file from the motor refinement was used as a template for re-extraction to include portal density. The same protocol of particle subtraction was employed, using a mask to encompass the whole portal / motor complex. Subtracted particles were then refined with limited angular searches of 1.8 • . After postprocessing the resolution of the portal / large terminase complex reached 7.4 Å (Supplementary Figure S1C). The portal / motor complex was then reextracted to include the entire prohead and subject to symmetry expansion in C 12 . Particles were subject to 3D classification without alignment and the best fiv e classes selected. Duplicates were removed leaving 8354 particles for 3D refinement (Supplementary Figure S1C). The resolution of the complete packaging complex was 8.3 Å (Supplementary Figure S1C).

Model building
Portal and prohead atomic models were built in Coot 0.8.9.3 ( 56 ) and refined using Phenix 1.19 Cryo EM Real Space Refinement ( 57 ). PDB model 3E8K ( 46 ) was used as a starting model for the asymmetric prohead unit. Residues 130-382 wer e r efined for each of the 7 chains. The first 103 r esidues ar e cleaved off in the transition from Prohead I, and density for residues 104-129 was not sufficiently defined for model building suggesting flexibility ( 58 ). For the portal protein, a starting model for a single chain was established using an AlphaFold prediction ( 59 ) using uniprot reference P49859. This was symmetrised 12-fold, and residues 32-398 refined into the C 12 map. Model validation parameters are summarised in Supplementary Supplementary  Table S2.

Stalling and stabilising the DNA packaging motor
In vitro , the HK97 DNA packaging motor has been shown to stall at a low ATP concentration if an internal cos site is incorporated into the DNA substrate ( 45 ). This stalling behaviour was deemed desirable for structural characterisation, potentially allowing the motor to be 'fixed' in a single conformation, pre v enting unsynchronised packaging and motor detachment. Thus, DNA protection assays were utilised to examine optimal stalling conditions: small terminase was critical, and the ideal ATP concentration was pinpointed to 75 M (Figure 1 A). The optimised sample was then flooded with 5 mM ATP γ S and visualised by negati v e stain electron microscopy (Figure 1 B) to check for associated packaging motors. The 3D structure of stalled motor was deri v ed by cryo-electron microscopy (Figure 1 C) and single-particle analysis. An asymmetric reconstruction comprising prohead II and the portal / large terminase comple x, was deri v ed at 8.3 Å . Despite limited resolution, the reconstruction contained well defined density at the unique portal v erte x, likely corresponding to a large terminase oligomer. Local refinement of each protein constituent is described subsequently.

Structure of HK97 prohead II
The cryo-EM structure of the prohead was determined at 3.06 Å using icosahedral averaging (Figure 2 ). EM density (Figure 2 C) allowed for refinement of the previously determined 3.6 Å crystal structure ( 46 ). The prohead measures 550 Å in diameter and shows dislocated or 'skewed' trimers within the hexamers (Figure 2 A). This is consistent with structures of prohead II ( 46 ), depicting a stage of capsid ma tura tion prior to expansion and after cleavage of the scaffolding domains from the major capsid protein. This indica tes tha t proheads remain unexpanded during packaging and stalling. Importantly, unlike the earlier reported crystal structure ( 46 ) the present structure was deri v ed for proheads containing the packaging machinery, r epr esenting a biologically relevant form.

Structure of HK97 portal protein
The portal protein reconstruction shows clear 12-fold symmetry with an overall 'mushroom' shaped ar chitectur e (Figure 3 ). An atomic model was built into the 2.98 Å resolution map. The clip, stem, and crown domains line an extended DNA channel, with the wing domain spanning outwards (Figure 3 B). The diameter of the central channel varies from ∼35 Å at the crown domain, to ∼33 Å in the clip domain, and ∼22 Å in the tightest part of the stem (Figure 3 A). This is sufficiently wide for translocating Bform DNA ( 60 ). The local resolution of the portal protein (Figure 3 C) shows the wing domain particularly well re-solved, with lower resolution for the crown and especially the clip domain. A reconstruction of the portal protein from a separate sample of HK97 prohead II featuring the in situ portal protein, but without terminase (unpublished data), presents an informati v e comparison (Figure 3 D). While the global resolution of this 'empty prohead' portal is lower, at 4.1 Å , low-pass filtering each map to the same threshold indicates a distinct improvement in the definition of the clip domain, where density corresponding to ␣-helices can be clearly distinguished. Further differences appear also in the crown domain, which is extended in terminase absence.

Reconstruction of the packaging motor comprising the portal-large terminase complex
In spite of attempts to fix the large terminase motor in a single state --both by introducing a cos site in the DNA substrate and providing a high concentration of ATP γ S --processing of the da ta indica tes tha t the motor is highly flexible and heterogeneous. Icosahedral symmetry expansion of the capsid followed by focussed 3D classification produced two broad portal / large terminase classes. Both showed strong signal at the large terminase locus but little definition, indicati v e of fle xibility (Supplementary Figure S1B, C). Indeed, further 3D classification re v ealed a whole spectrum of conformation, only se v eral of which displayed defined subunits (Supplementary Figure S1C). Subsequently, the terminase oligomer reconstruction represents just  17 584 particles out of a total of a pproximatel y 82 279 portal / large terminase particles.
The asymmetric reconstruction, estimates at 8.8 Å resolution (Figure 4 A), allows the fiv e large terminase subunits to be clearly observed, which are arranged in a pentameric ring encircling the dsDNA substrate. This stoichiometry for large terminase is in keeping with pr evious fluor escent photob leaching e xperiments ( 45 ) and the motor assemblies of other dsDNA bacteriophages ( 2 , 4-6 , 40 ). When the same particles were re-extracted to encompass both the large terminase and the portal protein in complex, the resolution im-proved to 7.4 Å (Figure 4 B), consistent with a more structurally rigid conformation. Clearly resolved rod-like density for the DNA within the lumen of the large terminase appears to make contact with both the N-and C-terminus domains of large terminase monomers in both reconstructions (Figure 4 ).
The large terminase reconstruction is in good agreement with the crystal structure of the monomer ( 45 ) with densities for each ATPase and nuclease domain well resolved (Figure 4 A-C). Flexibility within the complex is likely confined to inter-domain and inter-subunit movement, as opposed to domain rearrangement, consistent with the globular nature of the two domains and the ability to crystallise single-domain constructs of related large terminases mor e r eadily than full-length constructs (61)(62)(63)(64)(65)(66)(67). Thus, individual domains were subject to rigid-body fitting into the density ( 57 ) (Figure 4 D). The more compact nuclease domains were fitted into the ring directly below the portal, with the N-terminal ATPase domains positioned more flexibly beneath, with ∼25 bp DNA spanning the central tunnel of the terminase assembly.

Motor assembly induces asymmetry in the portal protein
The density corresponding to the portal clip domain within the packaging complex, compared to the portal clip domain in the empty prohead, is poorly resolved. This suggests distortion away from C 12 symmetry during packaging. We hypothesise that the plasticity of the clip domain compensates for the symmetry mismatch at the portal-large terminase interface and facilitates interactions with the large terminase. Meanwhile, the reduced height of the crown domain in the portal / large terminase complex echoes structural changes in related portal proteins, where the mounting pr essur e from DN A within the ca psid is thought to induce portal compression ( 34 , 37 ). Flexibility within each of these domains has been indicated in studies of other viruses during DNA packaging ( 37 , 68 ).

Small terminase likely dissociates after inducing large terminase endonuclease activity
Small terminase is essential for the stalling of the HK97 large terminase motor, and thus present in the sample, but no obvious density corresponding to a small terminase is apparent within the density maps. The nonameric ring of small terminase has a molecular weight of 160 kDa, which should be discernib le e v en if positioned fle xib ly. This indicates a transient role for small terminase in the complex forma tion. We hypothesise tha t the small terminase remains bound to this downstream cos site throughout packaging, acting as a roadblock for incoming large terminase, and instigating the conformational change r equir ed to switch the motor from displaying ATPase activity to endonuclease activity. In turn, small terminase may dissociate from the DNA (Figure 5 C). The documented stimulatory effect of the small terminase ( 45 ), in this model, would then be attributed solely to recruitment of large terminase to the viral DNA, thus enhancing the number of packaging initiation e v ents.

DNA cleavage at the large terminase-portal interface mimics termination
One striking feature of the portal-large terminase map is the apparent lack of DNA density within the portal channel (Figure 4 C). DNA extends through the lumen formed by the large terminase oligomer and stops at the interface between the large terminase and portal. The length of this  DNA is consistent with approximately 25 bp of B-form DNA. Packaging assays consistently show protected DNA within proheads of ∼1.3 kbp, suggesting that the motor has packaged from the 5 end of the DNA substrate and paused at the cos site (Figure 1 A).
This conformation mimics the packaging termination sta te and indica tes tha t on reaching a cos site after packaging one complete genome unit, large terminase endonuclease activity was activated. The nuclease domain is expected to employ the two-metal catalysis mechanism, as it adopts the classical RNase H family fold, with closest similarity to the RuvC endonucleases ( 62 , 63 , 65 , 69 ). Docking fiv e copies of the HK97 large terminase crystal structure into the oligomeric density (Figure 4 D) shows that each subunit is oriented with the nuclease acti v e site exposed towards the cleaved DNA end. These structural observations are consistent with the observed cut in the DNA and the termina tion sta te of the motor.
In viv o , DNA cleava ge is expected to trigger dissociation of the motor allowing for tail attachment ( 70 ), Figure 5 B. In our sample howe v er, large terminase remains bound to the portal (Figure 5 C). This suggests that packaging termination may occur via two distinct steps, controlled by different signals: (i) DNA cleavage at a cos site is mediated by small terminase large terminase interactions; (ii) release of the large terminase occurs in response to headful pr essur e signalling, likely relayed through the portal. In the structure of prohead II, presented here, the capsid remains relati v ely empty, and unexpanded, as the ∼1.3 kb length of DNA inside r epr esents just ∼3% of 39.7 kb genome. In the absence of the headful signalling the large terminase hence does not detach, b ut cleava ge at the cos site can still occur ( Figure 5 ).

T r anslocation is mediated by interdomain extensioncontraction
An asymmetric interaction between the large terminase and the portal clip domain is apparent in the portal-large terminase r econstruction (Figur e 4 ). Clear contact with the portal protein appears only between one large terminase subunit, with a second adjacent subunit showing limited interactions. The entire large terminase assembly is also tilted 10 • to the portal tunnel axis (Figure 4 A). Asymmetric alignment was similarly observed for the 29 motor, where the large terminase channel is tilted at 12.5 • to the portal axis ( 40 ). Howe v er, in contrast, the 29 portal-terminase interaction is mediated by a pRNA, which is absent in the HK97 system, while the terminase itself lacks nuclease activity ( 71 ).
In the docked crystal structure of the HK97 large terminase (Figure 4 D), the two subunits which contact the portal (depicted in red and or ange), are separ ated axially by a further ∼4.5 Å (or 1.5 bp of DNA), relati v e to the subunits shown in green and blue ( 72 ). Both domains of the extended subunits contact DNA (Figure 6 B), whilst the contracted subunits contact DNA via the nuclease domain only (Figure 6 B). Experimental data on multiple related large terminase has shown that ATP bound subunits show a high DNA binding affinity ( 7 , 73-75 ); and so, by analogy the extended HK97 subunits may be interpreted as ATP γ S bound whilst contracted subunits are more likely ADP bound. The presence of ADP in the sample would r equir e slow hydrolysis of ATP γ S, as observed in DNA translocases ( 76 ). Notably, the contracted subunits display broken contact with the clip domain of the portal protein, which could potentially facilitate a cyclically changing pattern of terminase-portal interactions throughout packaging. The final large terminase subunit, sho wn in yello w, displays a less dramatic extension of ∼2 Å , which could correspond to mixed occupancy of the nucleotide binding site (Figure 6 A) ( 76 ). An overlay of each docked large terminase ATPase domain after aligning docked nuclease domains, depicts the interdomain shifts around the pentameric ring (Figure 6 C). As such, contraction and relaxation of subunits is likely involved in DNA translocation -with ATP hydrolysis coupled to subunit contraction which pushes the DNA substrate into the prohead.
Contraction of large terminase subunits from an extended ATP bound state to a contracted ADP bound state has also been proposed as translocation mechanisms of the bacteriophages T4 and 29 ( 4 , 40 ). For T4, ATP hydr olysis is pr oposed to occur sequentially around the ring, so that only a single subunit is e v er present in the  'tense' contracted state ( 77 ). This does not entirely agree with the HK97 structure where subunits display a range of conformations (Figure 7 A). Meanwhile, the fiv e bacteriophage 29 ATPase domains are thought to make sequential shifts toward the vestigial nuclease domains (which remain in a planar ring throughout) (Figure 7 B). This completes a cracked helix to planar transition, in four ATP hydrolysis steps ( 40 ). Transposition of this mechanism onto our proposed HK97 model is also problematic since transition through full ADP occupancy could destabilise the terminase-portal contact.
An alternati v e comparison can be made with the DNA translocation mechanism proposed for the Esc heric hia coli translocase FtsK ( 76 ). In this mechanism, a molecular machine comprises six subunits, with each adopting a unique conformation with variable angles of separation between the ␣ and ␤ domains (as opposed to extension), Figure  7 C. Three subunits bound to ATP γ S, and a fourth with mixed ADP / ATP / APO occupancy, display DNA binding residues arranged in a spiral. The remaining two subunits are ADP-bound and do not engage with DNA ( 76 ). The proposed translocation mechanism for FtsK ne v er passes through a fully ADP bound state, with the rolling change in conformations around the ring shifting sequentially, akin to a spiral escalator ( 76 ). This mechanism is more consistent with the HK97 structure where subunits display a range of conformations and DNA is also engaged with the subunits in extended conformation, assigned as ATP γ S bound. A comparison between the individual monomers of HK97, 29 and FtsK ATPases (Figure 7 ) sho ws ho w each system depends on a varied extension of individual subunits mediated by inter domain mov ements. The comparison re v eals a significantly reduced range of contraction in the HK97 motor compared to 29. This again suggests that the HK97 motor may work akin the FtsK motor, with simultaneous conformational changes in all fiv e terminase subunits, propagating around the ring, so that each subunit in turn adopts e v ery conformation.
Nucleic Acids Research, 2023, Vol. 51, No. 13 7033 CONCLUSIONS Her e, we pr esent a structure of the complete packaging machinery of HK97 determined by cryo-EM. The highr esolution structur e of the prohead shows that the capsid adopts the immature, unexpanded state (prohead II), present at the initiation of DNA packaging. Howe v er, the well-defined DNA density within the large terminase pentamer ends abruptly at the portal protein interface, characteristic of DNA cleavage seen at packaging termination. This suggests that packaging termination r equir es two distinct signals, and while the cos sequence present in the DNA substra te is suf ficient to induce DNA cleav age b y the large terminase, headful pr essur e is r equir ed for motor dissociation. The cryo-EM structure of the portal protein, deri v ed at 2.98 Å resolution, shows that the clip domain alone deviates from the C 12 symmetry, facilitating interactions with the fiv e subunits of the large terminase. Docking of the large terminase subunits into the motor map suggests that the observed extension and contraction between the two domains of each subunit is involved in ATP-driven translocation of DNA into prohead, with both the N-and C-terminal subunits making contacts with DNA ( 55 ). This translocation model has commonalities with mechanisms proposed for the 29 and T4 packaging motors ( 40 , 77 ), as well as the DNA translocase Ftsk ( 76 ), each of which is proposed to utilise inter-subunit contraction to move DNA through the central pore of an oligomeric ring. The variable domain arrangement and the extent of observ ed contraction / e xtension of each subunit of FtsK coupled with the rolling ATP-ADP exchange, fits particularly well with the observed asymmetric nature of the HK97 large terminase pentamer, and the limited interaction with the portal protein.